PfGARP binding to enzyme-treated RBCs and GYPB-null cells. (A) PfGARP-binding assay of enzyme-treated RBCs. TRX–PfGARP-M binding is sensitive to chymotrypsin treatment (elution lane 4; white asterisk). Input of proteins (left side) and bound proteins (right side) are shown. Lanes 1 and 2 show TRX and TRX–PfGARP-M in untreated human RBCs, respectively. Lanes 3 through 5 correspond to TRX–PfGARP-M in RBCs treated with neuraminidase (5.0 units/mL), chymotrypsin (1.0 mg/mL), and trypsin (1.0 mg/mL), respectively. (B) Unbound excess TRX and TRX–PfGARP-M are shown at equivalent amounts in all groups recovered from the binding assays. (C) Binding of TRX–PfGARP-M to normal and GYPB-null human RBCs. Normal human RBCs were used as positive control to demonstrate TRX–PfGARP-M binding. TRX–PfGARP-M binding to GYPB-null RBCs was similar to normal RBCs. In fact, a slight enhancement of TRX–PfGARP-M binding to GYPB-null RBCs was observed in several experiments. No binding was detected with the negative control (TRX). (D) Substantial enhancement of TRX–PfGARP-M binding toward chymotrypsin-treated GYPB-null human RBCs was detected. GC, GYPB-null chymotrypsin-treated human RBCs; GU, GYPB-null untreated human RBCs; NU, normal untreated.